In our studies of stuttering, we have been working with a group of Pakistani stuttering families in which we have previously shown that a gene located on chromosome 12 is implicated in the cause of this disorder. In the past year, we have identified mutations in the GNPTAB gene at this locus that occur in individuals who stutter but not in normal controls. Based on these results, we have identified mutations in two genes that function in the same biochemical pathway, GNPTG and NAGPA. Together these results indicate that stuttering can be associated deficits in the lysosomal targeting pathway, In the past year we have performed additional family ascertainment and recruitment in Pakistan. This has resulted in the enrollment of six new consanguineous families, all characterized by a high density of individuals with persistent stuttering. Linkage analysis in these families is underway, and we have preliminary evidence for a variant gene located on chromosome 3 as the cause of stuttering in one of these families. We have also continued our studies in a group of families in Cameroon, West Africa, in which stuttering occurs consistent with a simple inherited trait. We have previously obtained evidence that a gene on chromosome 1 is responsible in the largest of these families, but follow-up genotyping in this region, plus a subsequent new genome-wide linkage analysis, performed with the next generation of single nucleotide polymorphism (SNP) markers, failed to confirm this linkage. We have now identified significant linkage on chromosome 15, and we are working to refine this linkage location and identify candidate genes in this region. Our studies of deficits in the sense of taste currently focus on sweet taste. During the past year, we have continued to test human subjects for their sensitivity to a variety of sugars and artificial sweeteners, and obtaining DNA from these subjects to identify the forms of the sweet taste receptor gene they carry. The goal of these studies is to identify the genetic basis of differences in sweet taste perception between different individuals. We have made psychophysical measurements of sucrose sensitivity of 165 unrelated normal subjects, and shown that individuals are stable in the ability to detect this sugar, and that there is wide variation among individuals in their sweet perception worldwide. We have obtained DNA sequence information from the T1R2 and T1R3 genes (which together encode the major sweet receptor) in these subjects, and have discovered that two SNPs upstream of the T1R3 coding sequence exert a significant effect on sucrose sensitivity in these subjects. These SNPs reside within the core consensus sequences for transcription factor binding sites, and together they account for approximately 16% of the variation in sucrose perception in our subject population. Using cell-based reporter assays, we have shown that these variants act by altering the transcription level of the T1R3 gene. We also showed that the frequency of the alleles associated with reduced sweet taste perception differ dramatically in different worldwide populations, with the highest frequencies occurring in sub-Saharan African populations, and the lowest frequencies occurring in western European populations. We are currently evaluating variation in other genes that encode known components of the mammalian taste transduction system. For these experiments, we are studying the same 165 normal individuals with well-characterized sweet taste perception measures. Variation in most of the candidate genes we've evaluated shows no evidence of association with variation in sweet taste perception in our subject population. However, we've obtained preliminary evidence that genetic variation in the G-alpha gustducin gene exerts a major effect on sweet perception, and further experiments to confirm this association, and to understand thee mechanism by which this variation exerts its effect, are underway.